The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.
Bibliographical noteFunding Information:
We acknowledge the High-throughput Sequencing Unit (head Stephan Wolf) of the Genomics and Proteomics Core Facility of the German Cancer Research Center (head Stephan Wiemann) for their assistance with the high-throughput sequencing, and the Omics and Data Management Core Facility (heads Jürgen Eils, Christian Lawerenz and Ivo Buchhalter) for the help with processing and management of the sequencing data. We are thankful to Christa Winkelmann (Institute of Pathology, Erlangen) for excellent performance of immunostainings, Cansu Cirzi for the help with ChIP-mentation libraries and Oliver Mücke for his help with MassARRAY. We thank ActiveMotif for providing a novel specific antibody against H3.3-G34W, as well as JPT (Pavlo Holenya and Ulf Reimer) for kindly providing a histone peptide array for antibody validation. We thank Stephan Pfister and David Jones for the permission to use PedGBM cohort, and Ana Banito for a thoughtful feedback about the manuscript. This work was in part supported by Funds from the Helmholtz Association (C.P.), the Korean National Cancer Center (Grant No. 1810050-1 to A.M.L.), the Korean National Research Fund (Grant No. 2017R1E1A1A01073670 to A.M.L.), Oxford NIHR BRC (U.O.), Arthritis Research UK (program grant 20522 to U.O.). P.L. and A.B. are DKFZ Postdoctoral Fellows. P.L. was supported by the Helmholtz Association AMPro Project (ZT00026 to C.P.). A.K., A.M. T., and D.M. are fellows of the Helmholtz International Graduate School for Cancer Research, J.H. is fellow of the German-Israeli Helmholtz Research School in Cancer Biology, C.J. is a recipient of a PhD fellowship from the Royal Commission for the Exhibition 1851, China-Oxford Scholarship Fund and UCB-Oxford Fellowship.
© 2020, The Author(s).